Anti-proliferative potential and oxidative reactivity of thermo-oxidative degradation products of stigmasterol and stigmasteryl esters for human intestinal cells

Stigmasterol in free and esterified form is incorporated in LDL cholesterol-lowering food products, intended for direct consumption and cooking, baking, and frying. Under thermal treatment, stigmasterol compounds may constitute a source of thermo-oxidative degradation products and oxyderivatives with potentially adverse health effects. This study aimed to analyze the anti-proliferative potential and genotoxicity of thermo-oxidatively treated stigmasterol (ST), stigmasteryl linoleate (ST-LA), and oleate (ST-OA). The effects on cell viability and proliferation, cell cycle progression, intracellular reactive oxygen species (ROS) generation, and DNA damage were analyzed in normal human intestinal cells. The mutagenic potential was assessed in a bacterial reverse mutation test using Salmonella enterica serovar Typhimurium strains involving metabolic activation. Stigmasteryl esters showed a significantly lower potential to affect intestinal cell viability and proliferation than non-esterified ST, regardless of heating. Thermo-oxidatively treated ST suppressed intestinal cell proliferation by arresting the cell cycle in the G2/M phase and DNA synthesis inhibition. The enhanced intracellular ROS generation and caspase 3/7 activity suggest targeting intestinal cells to the apoptosis pathway. Also, heated ST-LA intensified ROS production and elicited pro-apoptotic effects. Thermo-oxidative derivatives of ST and ST-LA may evoke harmful gastrointestinal effects due to their high oxidative reactivity towards intestinal cells.

The effect of stigmasterol and its esters on DNA synthesis in normal colon mucosa cells. The treatment of the CCD841 CoN cells with ST affected DNA synthesis substantially. The amount of BrdU incorporated into newly synthesized DNA decreased dose-dependently in the cells treated with non-heated ST (Fig. 3a). ST at 5 μg/mL concentration was the first dose that significantly inhibited DNA synthesis (↓10%, P = 0.028). ST at the maximum concentration tested (40 μg/mL) decreased DNA synthesis by 61% (Fig. 3a). ST thermal treatment lowered ST inhibitory potential. The heated ST disturbed DNA synthesis when the highest concentrations of 20 μg/mL and 40 μg/mL were applied, reducing BrdU incorporation in newly synthesized DNA by 31% and 39% at these doses (Fig. 3c). DNA synthesis inhibition was not observed after treating normal colon mucosa cells with esters ST-LA and ST-OA, regardless of their thermal processing (Fig. 3b,d).

Effect of stigmasterol and its esters on cell cycle progression and apoptosis. To investigate how
ST, ST-LA, ST-OA, and their thermal degradation products can influence the growth of human normal colon mucosa CCD841 CoN cells, the cell cycle was analyzed by flow cytometry. Cell distribution in different cell cycle phases is presented in Fig. 4a. The results indicate that the compounds at the maximum dose analyzed (40 μg/ mL) interfered with the progression of the normal cell cycle of CCD841 CoN cells. The non-heated and heated ST induced significant cell accumulation in the G 2 /M phases. Moreover, ST subjected to the thermal process caused an increase in the subpopulation of dead cells with lower DNA content and decreased the G 0 /G 1 phase cell population. Slight expanding the cell subpopulation with reduced DNA content was also observed in the cultures treated with stigmasteryl esters (ST-LA and ST-OA) at the maximum dose of 40 μg/mL, indicating their cell death-inducing effect, regardless of heating (Fig. 4a). www.nature.com/scientificreports/ Moreover, treatment with test compounds induced caspase-3/7 activation in colon mucosa cells. The highest activation of caspase-3/7 was observed in cells exposed to ST (8.7-fold) and ST-LA (7.3-fold) after heat treatment (Fig. 4b). Also, exposure of cells to heated ST and ST-LA enhanced intracellular ROS production, which increased by 21% and 34%, respectively (Fig. 4c).

Effect of stigmasterol and its esters on DNA damage in normal colon mucosa cells. Data
obtained from comet assay indicates that ST and its esters at the highest concentration (40 μg/mL) did not induce extensive DNA strand breaks in the colon mucosa cells. Slight DNA damage was shown by classifying comets according to the range of % DNA in the tail (Fig. 5a). Only two comet classes: no damage and low damage (class 0 and 1), were identified in the control cell population. Cell treatment with non-heated or heated ST resulted in the appearance of comets category 2 characterized by medium DNA damage with DNA content in the tails ranging from 25 to 45%. The comets category 2 constituted 3.5% and 3.8% of the populations treated with non-heated or heated ST, respectively. Also, ST-LA and ST-OA caused medium DNA damage, mainly when thermally processed esters were applied. Cell populations with medium DNA damage were estimated at 8.5% and 5.9%, respectively, following treatment with ST-LA and ST-OA (Fig. 5a). Moreover, colon mucosa cells with high DNA damage (DNA content of 45-70% in comet tails) accounted for 1-2% of the cell population exposed to ST esters, regardless of their heat treatment. Although few comets with high damage DNA content were observed, the total comet score (TCS) showed no significant differences in the comet class distribution after cell exposition to both non-heated and heated ST, ST-LA, and ST-OA. Very high DNA damage (> 70% DNA in comet tail) was identified only in cells exposed to 100 μM H 2 O 2 used as reference oxidant (Fig. 5b), which was reflected in high TCS value (263.1 ± 29.4) (Fig. 5b).
Mutagenic activity of stigmasterol and its esters. The strain S. typhimurium His -TA102, which is sensitive to a variety of oxidative mutagens 29,30 , was applied to detect mutagenic activity associated with the oxidative potential of ST, its esters (ST-LA and ST-OA), and their thermal degradation products. Tert-butyl-H 2 O 2 used as a referent oxidant caused a significant response in the TA102 strain as indicated by the mutagenic activity calculated at 7.9 and 2.5, without and with the metabolic activation, respectively ( Table 1)  A positive mutagenic response was observed in the S. typhimurium TA100 strain following exposure to the non-heated and heated ST without metabolic activation (Fig. 6d). The mutagenic index values were determined at 1.9 and 2.2 levels, respectively. ST also affected the frequency of mutations in the TA98 strain with microsomal fraction supplementation (Fig. 6g). The number of revertant colonies increased 1.6-fold and 2.5-fold in the TA98 strain treated with non-heated and heated ST, respectively. In contrast, ST esters (ST-LA and ST-OA), regardless of heating treatment, did not induce the reversion of mutations in TA100 and TA98 tester strains in the presence and absence of the microsomal fraction (Fig. 6e,f,h,i).

Discussion
Functional food enriched in ST and its esters is intended for direct consumption and recommended for cooking, baking, and frying. After thermal treatment, it may constitute a source of thermo-oxidative degradation products, including ST oxidation derivatives and low-molecular-weight compounds, such as volatiles and oligomers 6 , with not well-documented bioavailability, safety, and biological potential to human cells and tissues 19 . Previously reported research showed that thermo-oxidative treatment of ST and its esters (ST-LA and ST-OA) at 180 °C for 8 h causes extensive sterol and fatty acid moiety degradation. Heating produced significant amounts of the ST oxidation products (SOPs) and degradation derivatives, including ST oxides, polar dimers, trimers, other oligomers, and non-polar dimers. ST, ST-OA, and ST-LA degradation products' and SOPs' profiles are shown in the article published previously 25 .
This study assessed cytotoxicity, genotoxicity, and mutagenicity of thermo-oxidatively treated ST, ST-OA, and ST-LA using biological in vitro models. Cytotoxicity analyses: MTT test 25 and MultiTox-Fluor Multiplex Cytotoxicity Assay indicated the relatively high cytotoxic potential of non-esterified ST. The half-maximal effective concentration (EC 50 ) of free ST, which caused a 50% decrease in colon mucosa CCD 841 CoN cell viability, was calculated at 2.95 μg/mL 25   www.nature.com/scientificreports/ treatment with ST in a concentration as low as 1 μg/mL. It has also been shown that free ST at higher doses of 5-40 μg/mL significantly inhibits DNA synthesis in the colon mucosa cells, exerting potent antiproliferative effects. In the literature, ST inhibitory effects were also reported in other human normal cells, including small intestine FHs 74 Int and liver epithelial THLE-2 cells 25 , and human umbilical vein endothelial cells (HUVECs) and iPSC-derived cardiomyocytes 31 . ST was found to affect the viability of cardiovascular-relevant cell models highly. Besides, the harmful cardiac phenotype was observed in stigmasterolemic mice, indicating that ST is a potentially toxic compound 31 .
The experiments performed within this work indicate that ST, independently of thermal treatment, suppressed the proliferation of colon mucosa cells by inhibiting DNA synthesis, arresting the cell cycle in the G 2 /M phases, and targeting cells to the apoptosis pathway. ST anti-proliferative effects were analyzed previously in cancer cell research, which showed that ST can interact with various cellular targets and pathways. For instance, ST has been found to suppress gastric cancer cell proliferation via G 2 /M phase cell cycle arrest and apoptosis induction 32   Among SOPs, 7β-OH-ST, epoxydiol, diepoxide, and triol-ST were identified as the most cytotoxic to the human monocytic U937 cells 21 . The increased SOPs content in ST subjected to the thermo-oxidation process affected the enhanced oxidative reactivity and ST capacity to elevate the intracellular ROS level in treated colon mucosa CCD 841 CoN cells. Excessive ROS accumulation may drive cells into the apoptosis pathway. ROS at low and moderate doses regulate normal physiological functions involved in cell cycle progression and proliferation, cell differentiation, migration, and cell death. While excessive ROS accumulation causes oxidative damage to cellular macromolecules (proteins, lipids, DNA), membranes, and organelles, which may induce apoptotic cell death 33 . The effect of SOPs on intracellular ROS production and ROS-activated apoptosis in normal human cells has not been evidenced previously. However, several studies have suggested a crucial role of oxidative stress in apoptosis induced by COPs 34,35 . In addition, some reports indicated that the cytotoxicity of COPs and POPs is related to superoxide anion generation and lipid peroxidation 36 , the excessive accumulation of which may promote apoptosis 37 . Apoptosis promotion in the colon mucosa CCD 841 CoN cells treated with ST was manifested by significantly increased caspase 3/7 activity, one of the critical effector caspases involved in the final execution of dying cells. Boosted caspase 3/7 activity was detected in the cells exposed to heated and non-heated ST, although the non-heated ST did not induce enhanced intracellular ROS production. The ST auto-oxidation process and the formation of oxyderivatives, including 7keto-ST and 7βOH-ST 25 , can explain this phenomenon. The SOPs level in unheated ST samples could be too low to enhance ROS generation but high enough to mediate signal transduction pathways. Furthermore, it was found that both non-heated and heated ST caused a slight increase in the cell population with medium DNA damage, which may suggest its potential genotoxicity independent of the thermo-oxidative transformations.
The slight genotoxic effect detected in ST-treated colon mucosa cells was the reason for analyzing the ST mutagenic and pro-mutagenic potential using three mutated Salmonella typhimurium TA100, TA102, and TA98 strains. The Salmonella tester strains harbor different mutations: hisD3052 (TA98 strain), hisG46 (TA100 strain), and hisG428 (TA102 strain) in the genes of the histidine operon. Cells of the tested strains have also modifications enhancing their sensitivity to mutagenic conditions 30 . The experiments determined the pro-mutagenic activity of tested compounds through their metabolic activation in the presence of Aroclor 1254-induced rat liver microsomal fraction. Non-heated ST showed the ability to reverse mutations in the strain TA100 and strain TA98 only under metabolic activation. However, the mutagenic indexes not exceeding 2.0 in values, as required for mutagenic compounds, did not indicate ST mutagenic potential. For comparison, the mutagenic index of 2-aminofluorene and sodium azide, applied as reference mutagens for TA98 and TA100 strains, was determined at 9.8 and 13.6, respectively. Therefore, the non-heated ST was not considered mutagenic. More significant effects were detected in the Salmonella strains induced by ST subjected to thermo-oxidative treatment. Interestingly, the heated ST increased the number of revertants in the TA102 strain sensitive to various oxidative mutagens 30 , indicating the pro-oxidative capacity of ST derivatives produced during the thermo-oxidative process. SOPs generated when ST was heated at 180 °C were likely responsible for the oxidative mutagenicity detected in the TA102 strain. In contrast, the mutation reversal was not observed in the TA102 strain treated by heated ST under conditions stimulating metabolic bioconversions. These findings indicate that natural metabolic and antioxidant systems may limit the mutagenic activity of oxidant SOPs during the thermal proceedings. Moreover, considering the low mutagenic index (< 2.0), the oxidative mutation risk is probably relatively low regardless of metabolic activation. However, a significant increase in the mutation frequency was observed in the TA98 strain treated with heated ST under metabolic activation. In this experiment, the mutagenic index was determined at 2.5, indicating the potential pro-mutagenicity of ST after thermal treatment and metabolic transformation.
Previous studies have shown that free ST is degraded rapidly, generating a diverse group of thermo-oxidative degradation products and oxyderivatives 21,25 which may exhibit significant oxidative reactivity and induce cytotoxic and genotoxic effects in normal human cells as demonstrated in the presented studies. It was found that ST esterification with oleic and linoleic acids limited ST degradation and the formation of oxidized sterols, Table 1. Mutagenic index calculated for reference mutagens applied as positive controls to induce mutation in tester strains without or with metabolic activation (−/+ S9).

Reference mutagen
Metabolic activation  Previous studies have shown that the esterification of ST with oleic and linoleic acids impedes the ST degradation and formation of oxidized sterols, degradation products, and oligomers during thermo-oxidation treatment 25 . Limiting the formation of thermo-oxidative ST derivatives-compounds of unknown bioavailability and non-defined impacts on human cells and tissues is an important issue for maintaining the safety of ST-enriched food products, especially during cooking and processing. ST-LA and ST-OA stigmasteryl esters differ in forming degradation products and oxyderivatives during thermo-oxidative treatment and, thus, also in cytotoxicity and oxidative reactivity. ST-OA is characterized by higher toxicological safety; independently of thermal treatment, it does not induce cytotoxic and genotoxic effects nor elevate the intracellular ROS level in normal human colon cells. In contrast to ST-OA, the heating causes enhanced ST-LA oxidative reactivity and increases its cytotoxic potential in the intestinal cells. The findings suggest that ST-OA may constitute a non-cytotoxic ST compound to form special-purpose functional foods intended for direct consumption and the thermal proceeding. However, further preclinical studies should be continued to determine ST-OA bioavailability and functionality and exclude unfavorable health effects, such as pro-atherogenicity or pro-inflammatory.

Materials and methods
Preparation of stigmasteryl esters. Stigmasterol-ST (≥ 95%), oleic acid-OA (≥ 99%), and linoleic acid-LA (≥ 99%) standards were purchased from Sigma-Aldrich (St. Louis, MO, USA). ST-OA and ST-LA were obtained by chemical esterification based on the Neises and Steglich method 42 according to the protocol described by Kasprzak et al. 25 . ST (500 mg) dissolved in dichloromethane (30 mL) was placed in a three-necked flask. The air in the flask was replaced with argon. The catalyst: N,N′-Dicyclohexylcarbodiimide (500 mg) and 4-Dimethylaminopyridine (15 mg), and fatty acid (OA or LA) (600 mg) were then added to the flask. Esterification was performed at room temperature for 24 h in the dark. The reaction mixture was transferred to a separatory funnel and extracted in triplicate with distilled water (10 mL), with lower layers collecting. The fractions collected were concentrated under a vacuum at 30 °C, and the residue was dissolved in hexane (20 mL). The esterification mixture was purified on a silica gel column (45 × 2.5 cm). The ester fraction was eluted with hexane:ethyl acetate (9:1) mixture (450 mL). TLC was used to check the ester fraction purity. www.nature.com/scientificreports/ esters were analyzed to detect degradation products and oxyderivatives. Analytical methods were described in the article published previously 25 . Table S1 presents  Cell cycle analysis. CCD 841 CoN cells were grown in 6-well plates at an initial cell density of 2 × 10 4 cells/ cm 2 for 24 h under standard culture conditions. The cells were treated for 24 h with the analyzed compounds at a concentration of 40 μg/mL; the high dose was applied because of the relatively low cytotoxic potential of ST-OA and ST-LA as determined in cytotoxicity studies. In addition, the cells were exposed to 0.05 μM camptothecin (Sigma-Aldrich) as a positive reference compound known to modulate cell cycle progression.
After treatment, the cells were harvested by trypsinization, washed in PBS, and fixed in 70% ethanol. Then, the cells were stained with 50 μg/mL propidium iodide in the presence of 100 μg/mL RNase (Sigma-Aldrich). The sample preparation method and staining protocol were described previously 43 . The cycle phase distribution was analyzed with an Amnis™ FlowSight™ flow cytometer (Luminex Corporation, TX, USA). Caspase 3/7 activity assay. CCD 841 CoN cells were grown in black 96-well plates at an initial cell density of 2 × 10 4 cells/cm 2 for 24 h under standard culture conditions. The cell cultures were treated for 24 h with ST, ST-OA, and ST-LA at a 40 μg/mL dose. After treatment, caspase-3/7 activity was determined using the APO-One Homogeneous Caspase-3/7 Assay (Promega Corporation, Wisconsin, USA), which is based on the proteolytic cleavage of C-terminal side of aspartate residue in the DEVD peptide substrate by caspase 3/7 into fluorescent rhodamine 110 (R110). Analysis of caspase 3/7 activity was carried out following the manufacturer's instructions. Briefly, the cells after the treatment were incubated at room temperature with Apo-ONE ® Caspase-3/7 Reagent-bifunctional cell lysis/caspase activity buffer combined with the pro-fluorescent caspase-3/7 substrate Z-DEVD-R110. After 4-h incubation, fluorescence was measured at an excitation wavelength of 485 nm and an emission wavelength of 530 nm using a Tecan M200 Infinite microplate reader. Data obtained were normalized to cellular protein content, quantified by BCA assay (Pierce ® BCA Protein Assay Kit, Thermo Scientific Inc., USA) according to the manufacturer's protocol. Intracellular ROS measurement. CCD 841 CoN cells were grown in 6-well plates at an initial cell density of 2 × 10 4 cells/cm 2 for 24 h under standard culture conditions. The cell cultures were treated for 24 h with the analyzed compounds at a 40 μg/mL concentration. After treatment, the cells were harvested by trypsinization, washed with Hank's Balanced Salt Solution, and incubated with 10 μM 2′,7′-Dichlorodihydrofluorescein diac- DNA damage detection. The cells were grown at 6-well plates at the established density and standard culture conditions and exposed to the non-heated and heated ST, ST-OA, and ST-LA for 48 h. The non-treated cells and cells treated with H 2 O 2 (100 μM, 30 min) to induce oxidative DNA damage constituted the negative and positive controls, respectively. After treatment, cells were analyzed for DNA damage using a single cell gel electrophoresis (SCGE) called comet assay, described in detail previously 44 . Briefly, harvested cells suspended in low melting point agarose were put onto microscope slides pre-coated with normal melting point agarose and subjected sequentially to lysis, alkaline electrophoresis (pH > 13), and neutralization (pH 10). The cells were stained with SYBRGold (Molecular Probes), viewed under a fluorescence microscope (Axiovert 200, Zeiss, Carl Zeiss, Gottingen, Germany), and analyzed using CometScore™ software (TriTek Corp., Sumerduck, VA, USA). At least 100 cells were analyzed on a microscope slide; 300 cells were considered for DNA damage detection in one sample. Data were expressed as the mean percentage of DNA content in the comet tail. Moreover, depending on DNA content in comet tails, comets were classified into five categories: class 0 (< 1%, no damage), class 1 (1-25%; low damage), class 2 (> 25-45%; medium damage), class 3 (> 45-70%; high damage), class 4 (> 70%; very high damage). Based on the comets' categorization, the total comet score (TCS) was calculated according to the formula: TCS = 0(n) + 1(n) + 2(n) + 3(n) + 4(n), where "n" is the number of cells in each comet class (0-4) 45 .
Mutagenicity assay. Potential mutagenicity or pro-mutagenicity of non-heated and heated ST, ST-LA, and ST-OA was determined using the bacterial reverse mutation assay (Ames test) with Salmonella enterica subsp. enterica ser. typhimurium tester strains TA98, TA100, and TA102, which were obtained from the Polish Collection of Microorganisms of the Institute of Immunology and Experimental Therapy of the Polish Academy of Sciences in Wroclaw. Mutagenicity experiments were performed in the liquid pre-incubation assay, without and with metabolic activation by supplying Aroclor 1254-induced rat liver microsomal fraction (S9, Sigma-Aldrich) with NADP and cofactors for NADPH-supported oxidation, according to the procedure described by Mortelmans and Zeiger 39 . Briefly, reaction mixtures, consisting of 12-h bacterial culture, non-heated and heated compound (ST, ST-LA, ST-OA) at a concentration of 40 μg, 0.2 M phosphate buffer (pH 7.4), and alternatively S9 activating mixture, were pre-incubated at 37 °C for 20 min. The mutagens, 2-aminofluorene (100 μg), sodium azide (1 μg), and tert-butyl-hydrogen peroxide (tert-butyl-H 2 O 2 ) (50 μM) (all supplied by Sigma-Aldrich), were used as positive controls to reverse mutation in the TA98, TA100, and TA102 strain, respectively. The mutagen, 2-aminoanthracene (5 μg) (Sigma-Aldrich), was a positive control in pro-mutagenicity experiments. After pre-incubation, the reaction mixtures were added to the top agar supplemented with traces of histidine and biotin and poured onto the plates covered with minimum glucose agar. The cultures were incubated for 48 h at 37 °C, and the number of revertants (His+) colonies was counted manually.
The mutagenic index (MI), which reflects the number of induced revertants (Ri) with mutagen or compound tested divided by the number of spontaneously induced revertants (Rs), was applied to express mutagenic activity. Compounds with MI ≥ 2 can be recognized as potentially mutagenic 46 . The MI values calculated for reference mutagens applied to induce reverse mutation in tester strains ranged from 2.51 to 13.55 (Table 1).

Statistical analysis.
Data are presented as means ± SD from three independent replications. Statistical analysis was performed using STATISTICA version 13.3 software (Statsoft, Inc., Tulsa, OK, USA). A Student's t-test was used to compare two groups of data. One-way analysis of variance (ANOVA) followed by Tukey's post hoc test was performed to determine the differences between the mean values of multiple groups. P ≤ 0.05 was the cut-off point for a significant difference.

Data availability
The datasets generated during and/or analyzed during the current study are available from the corresponding author on reasonable request.